Liposomal particles, methods of making same and uses thereof

ABSTRACT

Liposomes termed as small unilamellar vesicles (SUVs), can be synthesized in the 20-50 nm size range, but encounter challenges such as instability and aggregation leading to inter-particle fusion. This limits their use as a therapeutic delivery agent. Increasing the surface negative charge of SUVs, via the attachment of anionic entities such as DNA/RNA, increases the colloidal stability of these vesicles. Additionally, the dense spherical arrangement and radial orientation of nucleic acids exhibits unique chemical and biological properties, unlike their linear counterparts. These liposomal particles, are non-toxic and though anionic, can efficiently enter cells without the aid of ancillary cationic transfection agents in a non-immunogenic fashion. These exceptional properties allow their use as delivery agents for gene regulation in different therapies and offer an alternative platform to metal core spherical nucleic acids.

This application is a Continuation of U.S. application Ser. No.16/242,704, filed Jan. 8, 2019, which is a Continuation of U.S.application Ser. No. 15/101,523, filed Jun. 3, 2016, which is a U.S.National Phase of International Patent Application No. PCT/US14/68429,filed Dec. 3, 2014, which claims the priority benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 61/911,334, filed Dec. 3,2013, and U.S. Provisional Application No. 61/982,269, filed Apr. 21,2014, the disclosures of which are incorporated herein by reference intheir entirety.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under HR0011-13-2-0018awarded by the Defense Advanced Research Project Agency and CA151880awarded by the National Institutes of Health. The government has certainrights in the invention.

SEQUENCE LISTING

This application contains, as a separate part of the disclosure, aSequence Listing in computer readable form (filename:2013-201_SeqListing.txt; Created: Dec. 3, 2014; 1,893 bytes), which isincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present disclosure relates to liposomal particles, methods of makingthe same, and uses thereof. Liposomal particles are useful in generegulation and drug delivery.

BACKGROUND

Chemistry has been explored to create liposomes and small unilamellarvesicles (SUVs). For example, Vogel et al., “DNA Controlled Assembly ofLipid Membranes,” U.S. Patent Publication Number 2010/0144848, disclosesthat DNA modified with two lipophilic anchors can form liposomes orSUVs. This post modification technique does not favor high surfacedensity modification.

Hook et el., “Oligonucleotides Related to Lipid Membrane Attachment,”U.S. Patent Publication Number 2013/0252852 describes liposomes or SUVscreated having an oligonucleotide having a first strand and a secondstrand of nucleic acid and two or more hydrophobic anchoring moietieslocated in its terminal ends, wherein the hydrophobic anchoring moietiesare found in the bilayer. Since two cholesterol molecules are used toanchor a molecule into the lipid bilayer, this post modificationtechnique does not favor high surface density modification.

Lu et al., “Amphiphilic Substances and Functionalized Lipid VesiclesIncluding the Same,” U.S. Patent Publication Number 2010/0166842describes liposomes or SUVs comprising at least two nucleotide segmentshybridized with each other. This non-post modification technique basedvesicle is less efficient in stabilizing vesicles since it incorporatesstabilizing moieties on both sides of the lipid bilayer.

Non-patent literature also reveals chemistry to create liposomes andSUVs, but each of these chemistries has its issues too. For example,“Liposome-Anchored Vascular Endothelial Growth Factor Aptamers”Bioconjugate Chem., 1998, 9, 573-582, describes the synthesis of aptamerDNA-functionalized liposomes and their application toward selectivecancer cell targeting. The liposomes created by this method averaged 80nanometers in size, had aptamer DNA molecules on both sides of thebilipid layer, and did not demonstrate gene regulation.

“Reversible Cell-Specific Drug Delivery with Aptamer-FunctionalizedLiposomes” Angew. Chem. Int. Ed. 2009, 48, 6494-6498, describes thesynthesis of aptamer DNA-functionalized liposomes and their applicationtoward selective cancer cell targeting and drug delivery. The liposomescreated by this method averaged between 140 nanometers and 200nanometers, utilize a cholesterol unit to anchor DNA into the lipidbilayer, comprise apatamer DNA molecules on both sides of the bilipidlayer, and did not exhibit gene regulation.

“Selective delivery of an anticancer drug with aptamer-functionalizedliposomes to breast cancer cells in vitro and in vivo” J. Mater. Chem.B, 2013, 1, 5288, discloses the synthesis of aptamer DNA-functionalizedliposomes and their application toward selective cancer cell targetingand drug delivery. This work is an extension of the research disclosedin “Reversible Cell-Specific Drug Delivery with Aptamer-FunctionalizedLiposomes” above Like before, these particles utilize a cholesterol unitto anchor DNA into the lipid bilayer, comprise aptamer DNA molecules onboth sides of the bilipid layer, and did not exhibit gene regulation.

The research in “Phospholipid Membranes Decorated by Cholesterol-BasedOligonucleotides as Soft Hybrid Nanostructures” J. Phys. Chem. B, 2008,112, 10942-10952, characterizes cholesterol DNA-functionalizedliposomes. In this report, liposomes of 33 to 35 nm in size wereprepared from 1-Palmitoyl-2-oleoylphosphatidylcholine (POPC) lipid andpost functionalized with cholesterol modified DNA molecule. This reportdoes not demonstrate gene regulation, and these particles utilize acholesterol unit to anchor DNA into the lipid bilayer.

“Bivalent Cholesterol-Based Coupling of Oligonucleotides to LipidMembrane Assemblies” J. Am. Chem. Soc. 2004, 126, 10224-10225, describesthe development of partially duplexed DNA strand containing twocholesterol units for anchoring into the lipid bilayer. The use of twocholesterol units to anchor a DNA strand into the lipid bilayer resultsin decreased surface density of oligonucleotides associated with theliposome.

In “Quantification of Oligonucleotide Modifications of Small UnilamellarLipid Vesicles” Anal. Chem. 2006, 78, 7493-7498, the researchersdescribe the development of a technique for the quantification of DNAstrands on a functionalized liposomal nanoparticle. The particledescribed comprises a partially duplexed DNA strand containing twocholesterol units for anchoring into the lipid bilayer. The use of twocholesterol units to anchor a DNA strand into the lipid bilayer resultsin decreased surface density of oligonucleotides associated with theliposome.

“Single-Molecule Detection and Mismatch Discrimination of Unlabeled DNATargets” Nano Lett. 2008, 8, 183-188, discloses 100 nanometer sizedliposomes functionalized with partially duplexed DNA strand containingtwo cholesterol units. This work is an extension of the researchdisclosed in “Bivalent Cholesterol-Based Coupling of Oligonucleotides toLipid Membrane Assemblies” and “Quantification of OligonucleotideModifications of Small Unilamellar Lipid Vesicles” above. Like before,these particles comprise a partially duplexed DNA strand containing twocholesterol units for anchoring into the lipid bilayer. The use of thetwo cholesterol units to anchor a DNA strand into the lipid bilayerresults in decreased surface density of oligonucleotides associated withthe liposome.

“DNA-Induced Programmable Fusion of Phospholipid Vesicles” J. Am. Chem.Soc. 2007, 129, 9584-9585, is an analytical paper on the fusion ofcholesterol DNA-functionalized liposomal nanoparticles. The vesiclesutilized in this paper were at least 100 nanometers in size.

“Determinants for Membrane Fusion Induced by Cholesterol-Modified DNAZippers” J. Phys. Chem. B, 2008, 112, 8264-8274, is an analytical paperon fusion of cholesterol DNA-functionalized liposomal nanoparticle, andis a continuation of the work from “DNA-Induced Programmable Fusion ofPhospholipid Vesicles” described above. This paper combines sequencespecific fusion with the utilization of a partially duplexed DNA strandcontaining two cholesterol units to anchor the oligonucleotide into thelipid bilayer (e.g., the partially duplexed DNA strand found in“Quantification of Oligonucleotide Modifications of Small UnilamellarLipid Vesicles” above).

“Liposome-Based Chemical barcodes for Single Molecule DNA DetectionUsing Imaging Mass Spectrometry” Nano Lett., 2010, 10, 732-737, is ananalytical paper on detection of specific DNA targets depending on theDNA sequence. This is an extension of the work from the same group thatreported “DNA-Induced Programmable Fusion of Phospholipid Vesicles” thatcombines sequence specific fusion with different DNA anchoring (usingbischolesteryl anchor, see: Anal. Chem. 2006, 78, 7493-7498).

“Programmable Assembly of DNA-Functionalized Liposomes by DNA” is ananalytical paper that discloses the assembly of cholesterol DNAfunctionalized liposomes. In this report, liposomes with a hydrodynamicdiameter of 114 and 251 nm were synthesized and post syntheticallyfunctionalized with cholesterol modified DNA molecules. The particles inthis report utilize cholesterol anchoring of the oligonucleotidemolecule into the lipid bilayer.

SUMMARY OF THE INVENTION

Liposomes are spherical, self-closed structures in a varying size rangeconsisting of one or several hydrophobic lipid bilayers with ahydrophilic core. The diameter of these lipid based carriers range from0.15-1 micrometers, which is significantly higher than an effectivetherapeutic range of 20-100 nanometers. Liposomes termed smallunilamellar vesicles (SUV), can be synthesized in the 20-50 nanometersize range, but encounter challenges such as instability and aggregationleading to inter-particle fusion. This inter-particle fusion limits theuse of SUVs in therapeutics.

To combat this instability, SUVs can be functionalized with polymers,peptides, DNA, and other molecules of interest by two distincttechniques. In a first approach, a modified molecule of interest isadded to the mixture of lipids, lipid film or hydration buffer duringthe synthesis of liposome. This approach results in a liposomescontaining a functional molecule of interest on both inner and outerlayers of the liposomal membrane. Generally speaking, structures createdby this method are not stable at a size smaller than 80 nanometers (nm).In an alternative approach, a SUV may be made by anchoring a substrateof interest into the lipid bilayer of the preformed vesicle (a “postmodification technique”). This alternative approach yields a liposomalnanoparticle containing a functional molecule of interest on the outerlayer of the liposomal membrane. Importantly, this alternative postmodification approach allows the creation of liposome of any size, evenless than 50 nanometers.

Accordingly, in one aspect the disclosure provides an architecturecomprising a lipophilic end and a non-lipophilic end. The lipophilicend, in some embodiments, comprises tocopherol. In additionalembodiments, the tocopherol is chosen from the group consisting ofalpha-tocopherol, beta-tocopherol, gamma-tocopherol anddelta-tocopherol.

The non-lipophilic end, in further embodiments, is a charged polymer. Insome embodiments, the charged polymer is an oligonucleotide. In relatedembodiments, the oligonucleotide comprises RNA or DNA, and in variousembodiments the RNA is an inhibitory RNA (RNAi) that performs aregulatory function. In still further embodiments, the RNAi is selectedfrom the group consisting of a small inhibitory RNA (siRNA), an RNA thatforms a triplex with double stranded DNA, and a ribozyme. In additionalembodiments, the RNA is a piwi-interacting RNA (piRNA), or the RNA is amicroRNA that performs a regulatory function. In some embodiments, theDNA is antisense-DNA.

In another aspect, the disclosure provides a method for making anarchitecture of the disclosure, the method comprising providing anoligonucleotide, providing a phosphoramidite-modified-tocopherol, andexposing said oligonucleotide to saidphosphoramidite-modified-tocopherol to make an architecture of thedisclosure.

In a further aspect, a liposomal particle is provided by the disclosure,said liposomal particle having a substantially spherical geometry, saidliposomal particle comprising a lipid bilayer comprising a plurality oflipid groups; and an oligonucleotide.

It is contemplated by the disclosure, in various embodiments, that saidplurality of lipid groups comprises a lipid selected from the groupconsisting of the phosphatidylcholine, phosphatidylglycerol, andphosphatidylethanolamine family of lipids.

In various embodiments, said lipid is selected from the group consistingof 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1,2-dimyristoyl-sn-phosphatidylcholine (DMPC),1-palmitoyl-2-oleoyl-sn-phosphatidylcholine (POPC),1,2-distearoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DSPG),1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DOPG),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), and1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DPPE).

In further embodiments, the oligonucleotide is an oligonucleotide-lipidconjugate containing a lipophilic tethered group, wherein saidlipophilic tethered group is adsorbed into the lipid bilayer. Thelipophilic tethered group comprises, in various embodiments, tocopherolor cholesterol.

The disclosure also contemplates that the tocopherol, in variousembodiments, is selected from the group consisting of a tocopherolderivative, alpha-tocopherol, beta-tocopherol, gamma-tocopherol anddelta-tocopherol. In yet further embodiments, the disclosure alsocontemplates that the lipophilic tethered group (i.e., the lipid anchor)comprises, for example and without limitation, palmitoyl, dipalmitoyl,stearyl, or distearyl.

The oligonucleotide, in further embodiments, comprises RNA or DNA. Inadditional embodiments, the RNA is a non-coding RNA, and in stillfurther embodiments, the non-coding RNA is an inhibitory RNA (RNAi). Thedisclosure further contemplates that, in some embodiments, the RNAi isselected from the group consisting of a small inhibitory RNA (siRNA), asingle-stranded RNA (ssRNA) that forms a triplex with double strandedDNA, and a ribozyme. In further embodiments, the RNA is a microRNA. Insome embodiments, the DNA is antisense-DNA.

In various embodiments, the diameter of said liposomal particle is lessthan or equal to about 50 nanometers. Regarding the surface density, thedisclosure provides compositions and methods wherein a liposomalparticle comprises from about 10 to about 100 oligonucleotides, or fromabout 10 to about 80 oligonucleotides. In some embodiments, the particlecomprises 70 oligonucleotides.

In some embodiments, the oligonucleotide is a modified oligonucleotide.

In another aspect of the disclosure, a method of making a liposomalparticle is provided, the method comprising adding a phospholipid to asolvent to form a first mixture, said first mixture comprising aplurality of liposomes; disrupting said plurality of liposomes to createa second mixture, said second mixture comprising a liposome and a smallunilamellar vesicle (SUV); isolating said SUV from said second mixture,said SUV having a particle size between about 20 nanometers and 50nanometers; and adding an oligonucleotide to the isolated SUV to makethe liposomal particle.

In some embodiments, the particle size of the plurality of liposomes insaid first mixture is between about 100 nanometers and 150 nanometers.In further embodiments, the particle size of the liposome and the SUV insaid second mixture is between about 20 nanometers and about 150nanometers. In still further embodiments, the liposomal particle has aparticle size less than or equal to about 50 nanometers.

In some embodiments, the oligonucleotide is an oligonucleotide-lipidconjugate containing a lipophilic tethered group, wherein saidlipophilic tethered group is adsorbed into the lipid bilayer. In relatedembodiments, the lipophilic tethered group comprises tocopherol orcholesterol. In further embodiments, tocopherol is chosen from the groupconsisting of a tocopherol derivative, alpha-tocopherol,beta-tocopherol, gamma-tocopherol and delta-tocopherol.

In further embodiments, the oligonucleotide comprises RNA or DNA. TheRNA, in some embodiments, is a non-coding RNA. In further embodiments,the non-coding RNA is an inhibitory RNA (RNAi). The disclosure furthercontemplates that, in additional embodiments, the RNAi is selected fromthe group consisting of a small inhibitory RNA (siRNA), asingle-stranded RNA (ssRNA) that forms a triplex with double strandedDNA, and a ribozyme.

In some embodiments, the RNA is a microRNA. In various embodiments, theDNA is antisense-DNA.

In some embodiments, the oligonucleotide is a modified oligonucleotide.

In another aspect of the disclosure, a method of inhibiting expressionof a gene is provided comprising the step of hybridizing apolynucleotide encoding said gene product with one or moreoligonucleotides complementary to all or a portion of saidpolynucleotide, said oligonucleotide being attached to the liposomalparticle of the disclosure, wherein hybridizing between saidpolynucleotide and said oligonucleotide occurs over a length of saidpolynucleotide with a degree of complementarity sufficient to inhibitexpression of said gene product.

In some embodiments, expression of said gene product is inhibited invivo. In further embodiments, expression of said gene product isinhibited in vitro.

In additional embodiments, the liposomal particle has a diameter aboutless than or equal to 50 nanometers. In some embodiments, theoligonucleotide comprises RNA or DNA. The RNA, in some embodiments, is anon-coding RNA. In related embodiments, the non-coding RNA is aninhibitory RNA (RNAi). The disclosure also contemplates that the RNAi,in various embodiments, is selected from the group consisting of a smallinhibitory RNA (siRNA), a single-stranded RNA (ssRNA) that forms atriplex with double stranded DNA, and a ribozyme. In some embodiments,the RNA is a microRNA. In further embodiments, the DNA is antisense-DNA.

In another aspect of the disclosure, a method for up-regulating activityof a toll-like receptor (TLR) is provided, comprising contacting a cellhaving the toll-like receptor with a liposomal particle of thedisclosure. In some embodiments, the oligonucleotide is a TLR agonist.In further embodiments, the toll-like receptor is chosen from the groupconsisting of toll-like receptor 1, toll-like receptor 2, toll-likereceptor 3, toll-like receptor 4, toll-like receptor 5, toll-likereceptor 6, toll-like receptor 7, toll-like receptor 8, toll-likereceptor 9, toll-like receptor 10, toll-like receptor 11, toll-likereceptor 12, and toll-like receptor 13.

In a further aspect, the disclosure provides a method fordown-regulating activity of a toll-like receptor (TLR), comprisingcontacting a cell having the toll-like receptor with a liposomalparticle of the disclosure. In some embodiments, the oligonucleotide isa TLR antagonist. In further embodiments, the toll-like receptor ischosen from the group consisting of toll-like receptor 1, toll-likereceptor 2, toll-like receptor 3, toll-like receptor 4, toll-likereceptor 5, toll-like receptor 6, toll-like receptor 7, toll-likereceptor 8, toll-like receptor 9, toll-like receptor 10, toll-likereceptor 11, toll-like receptor 12, and toll-like receptor 13.

The disclosure also contemplates that, in various embodiments, a methodas disclosed herein is performed in vitro. In further embodiments, thedisclosure contemplates that a method as disclosed herein is performedin vivo.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the synthesis of small unilamellar vesicles (SUV)functionalized with DNA or RNA on the surface of lipid vesicle. Thelarger size liposomes are sonicated into SUVs using a probe sonicator,and are separated from heavy impurities by ultracentrifugation.

FIG. 2 demonstrates the characterization of liposomal particles fromsmall unilamellar vesicles (SUVs). The dynamic light scattering (DLS)particle size data and transmission electron microscopy (TEM) pictureswere obtained before and after functionalization.

FIG. 3 demonstrates the stability of liposomal particles stabilized witholigonucleotides having different lipophilic ends to anchor theoligonucleotide to the liposome. Liposomes stabilized withtocopherol-modified oligonucleotides demonstrate better stability overbare liposomes, liposomes stabilized with cholesterol-modifiedoligonucleotides, and liposomes stabilized with stearyl-modifiedoligonucleotides. a) A gel electrophoresis image of FITC-encapsulatedSUVs that have been functionalized with oligonucleotides havingdifferent lipophilic ends; b) and c) Gel electrophoresis images ofFITC-encapsulated SUVs that have been functionalized with Cy5-labeledDNA.

FIG. 4 demonstrates that liposomal particles that have been stabilizedwith oligonucleotides have good temperature stability and show the rangeof tocopherol-modified DNA concentrations that were used to stabilizeSUVs. a) Stability of the liposomal SNA (LSNAs) after being stored at37° C. for 24 hours comparing to LSNAs that have been stored at 4° C. b)Gel electrophoresis showing the range of α-tocopherol modified DNAconcentrations that were used to stabilize the SUVs.

FIG. 5 comprises confocal images demonstrating that liposomal particlesdisclosed herein are able to enter cells. HeLa cells were treated withDNA (dT₃₀-Cy5 or dT₃₀) at a concentration of 100 nM in serum-free mediaand analyzed after 16 hours.

FIG. 6 shows cell viability assay data demonstrating that liposomesstabilized with tocopherol modified oligonucleotides do not exhibit asubstantial cytotoxic effect on cells in comparison to liposomes leftunmodified.

FIG. 7 depicts assembly of liposomal-spherical nucleic acids (SNAs) froma DOPC SUV and tocopherol-modified DNA.

FIG. 8 depicts stability studies of SUVs and LSNAs. (A) Dynamic lightscattering profile of SUVs after heating in buffer. (B) Dynamic lightscattering profile of LSNA after heating in buffer. (C) Schematicrepresentation of liposome decomposition in the presence of bovine serumalbumin, a major component of fetal bovine serum. (D) Degradation ofSUVs (upper trace) and LSNAs (lower trace) in the presence 10% fetalbovine serum, as monitored by the release of encapsulated rhodamine dye,which cause in increase in the fluorescence of the solution.

FIG. 9 shows (A) Melting transition of liposomal-SNA aggregatesmonitored as absorbance at 260 nm. (B) Absorbance spectra ofliposomal-SNAs before aggregation (lower trace) and after aggregation inthe presence of linker DNA strand (upper trace).

FIG. 10 shows (A) Confocal micrograph of SKOV3 cells incubated with 100nM Cy5-labelled liposomal-SNAs for 24 hours. Cell nuclei are stainedwith Hoechst 33342. (B) Cytotoxicity measurements of liposomal-SNAs andDharmaFECT-DNA complex in SKOV3 cells by MTT assay. (C) Cell uptake of5-Cy5-labelled DNA strand and 5′-Cy5-labelled liposomal-SNAs in SKOV3cells quantified by flow cytometry after a 1 hour (left bar in eachgroup) and 36 hours (right bar in each group) of incubation. (D) HER2gene knockdown in SKOV3 cells using anti-HER2 liposomal-SNA constructsat 1 μM DNA concentration.

FIG. 11 depicts a TEM micrograph of SUVs after isolation andpurification.

FIG. 12 depicts A) The equation used to calculate the total number ofliposomes in a given solution. Concentration of the lipid can bedetermined using ICP. For most of the studies described herein, workinglipid concentration 1.3 mM gives 1.361×10¹⁷ liposomes/L and the DNAloading of 71 DNA strands per particle (4 pmol cm²). B) Particlemobility of liposomal SNAs depending on the estimated oligonucleotideloading.

FIG. 13 shows the movement of FITC-encapsulated LSNAs that have beenfunctionalized with 5′-Cy5-labeled DNA strand on a 1% agarose gelelectrophoresis image. B) FITC channel showing movement of the liposomalcore on the gel due to the presence of negatively charged DNA corona. C)Cy5 channel indicates the difference in mobility due to size differencesbetween a free strand and those functionalized on the liposomalconstruct. Both channels co-localize on the same band.

FIG. 14 depicts the Ramos-Blue™ NF-κB/AP-1 reporter system.

FIG. 15 shows the activation of Ramos-Blue cells when exposed toCpG-containing oligonucleotides.

FIG. 16 is a graphical depiction of the synthesis of a liposomal SNA.

FIG. 17 shows a gel electrophoresis image of liposomal SNAs that weresurface functionalized with varying numbers of oligonucleotides. Theconcentration of 30 nm SUVs was 0.22 μM (determined by analysis ofphospholipid content by elemental analysis and approximation of 2.2×10³phospholipids per 30 nm SUV).

FIG. 18 shows the results of experiments in which liposomal particleswere used to knock down the expression of HIF1-α.

FIG. 19 shows the results of experiments in which liposomal particleswere used to knock down the expression of BAX.

DETAILED DESCRIPTION

Spherical nucleic acid (SNA) nanoparticle conjugates are structurestypically synthesized from inorganic nanoparticle templates and shellsof highly oriented nucleic acid ligands immobilized on the surface ofsuch particles [Mirkin et al., Nature 382: 607 (1996)]. SNAs have beenprepared in a variety of different forms [Cutler et al., J. Am. Chem.Soc. 134: 1376 (2012); Will et al., In Nanomaterials for Biomedicine;American Chemical Society: Vol. 1119, p 1-20 (2012)]. Core compositions,including gold, silica [Young et al., Nano Lett. 12: 3867 (2012)], ironoxide [Cutler et al., Nano Lett. 10: 1477 (2010); Zhang et al., Nat.Mater. 12: 741 (2013)], and Ag [Lee et al., Nano Lett. 7: 2112 (2007)]with shell compositions consisting of DNA, RNA, LNA [Seferos et al.,ChemBioChem 8: 1230 (2007)], and PNA [Lytton-Jean et al., AdvancedMaterials 21: 706 (2009)] have all been prepared and explored. HollowSNA structures consisting of cross-linked oligonucleotide [Cutler etal., J. Am. Chem. Soc. 133: 9254 (2011)] have been synthesized alongwith micelle-block copolymer structures [Li et al., Nano Lett. 4: 1055(2004); Alemdaroglu et al., Advanced Materials 20: 899 (2008); Liu etal., Chemistry—A European Journal 16: 3791 (2010); Chien et al., Chem.Commun. 47: 167 (2011)]. Although there is now a tremendous structuraland compositional diversity among the known SNAs, they all share somecommon properties and features. Their polyvalent architectures allowthem to cooperatively bind oligonucleotides and form duplex structuresthat exhibit very narrow melting transitions. These properties have beenexploited in the development of high sensitivity and high selectivitygenomic detection systems [Rosi et al., Chem. Rev. 105: 1547 (2005)].While linear nucleic acids do not enter cells well without polymer,peptide, or viral transfection agents, the three-dimensional SNAstructure is recognized by class A scavenger receptors [Patel et al.,Bioconjugate Chem. 21: 2250 (2010); Choi et al., Proc. Natl. Acad. Sci.U.S.A. 110: 7625 (2013)] and is rapidly taken into over 60 differentcell types without the need for an ancillary transfection agent[McAllister et al., J. Am. Chem. Soc. 124: 15198 (2002); Whitehead etal., Nat Rev Drug Discov 8: 129 (2009); Zhang et al., Biomaterials 31:1805 (2010)]. This property has made such structures important elementsin strategies for both intracellular detection [Zheng et al., Nano Lett.9: 3258 (2009); Prigodich et al., ACS Nano 3: 2147 (2009)] and generegulation via antisense or siRNA pathways [Rosi et al., Science 312:1027 (2006); Agbasi-Porter et al., Bioconjugate Chem. 17: 1178 (2006);Giljohann et al., J. Am. Chem. Soc. 131: 2072 (2009); Jensen et al.,Science Translational Medicine 5: 209ra152 (2013)].

The barrier to therapeutic use is high, however, especially when suchstructures are made from materials that have known problems withclearance or unknown biodistribution characteristics. Ideally, one wouldlike an SNA structure that is made from readily available startingmaterials, can be synthesized at scale, and consists of components thathave been a part of FDA approved pharmaceuticals [Cutler et al., J. Am.Chem. Soc. 134: 1376 (2012); Farokhzad et al., Drug Delivery Rev. 58:1456 (2006)]. Herein, a strategy for making such structures is provided,which consist of small liposomal cores stabilized with a dense shell ofa charged polymer with a hydrophobic tail that can intercalate betweenthe phospholipids that define the liposome structure. One such chargedpolymer contemplated for use is a nucleic acid. As with conventionalSNAs, these liposomal structures rapidly enter multiple cell lines andare used in some embodiments to effectively knockdown gene expressionvia antisense pathways. Conventional SNAs have been shown to enter cellsderived from many organs and tissues, including Breast (SKBR3,MDA-MB-231, AU-565), Brain (U87, LN229, U118), Bladder (HT-1376, 5637,T24), Colon (LS513), Cervix (HeLa, SiHa), Skin (C166, KB, MCF 10A),Kidney (MDCK), Brain (Rat Hippocampus Neurons, Astrocytes, Glial Cells),Bladder, Blood (PBMC, T-cells), Pancreas (Human (3-Islets), Skin(Human), Blood (Sup T1, Jurkat), Leukemia (K562), Liver (HepG2), Kidney(293T), Ovary (CHO), Fibroblast (NIH3T3), Macrophage (RAW264.7). Thespherical nucleic acid architecture facilitates the entry of theseconstructs into cells by binding to scavenger receptor A, acell-membrane receptor. A few non-limiting examples of cell lines whichexpress this receptor are HeLa, SKOV-3, U87, Neuro 2A, RAW cells, HepG2,Hep3B, MDA-MB-468, MCF-7, C8S, C166 Bend3, A549, Rab9, HeyA8, Jurkatcells.

The major drawback of employment of SUVs is their inherent instabilityin solution due to high propensity to fuse into bigger liposomalstructures. It is disclosed herein that functionalization of thesestructures with a dense layer of negatively charged DNA increases theirstability by, e.g., decreasing particle-particle interaction due to therepulsion of the negatively charged particle surfaces. In the course ofthe studies described herein, it was found that tocopherolfunctionalized DNA provides higher density of DNA strands on theparticle compared to other known hydrophobic DNA analogues,significantly increasing stability of the particle. In addition to thegeneral colloidal stability, high density of the DNA will increase theuptake of this nanoparticle via a scavenger receptor B pathway and willallow efficient delivery of the genetic material into a cell. Finally,the dense layer of DNA is expected to increase particle stability in abody, its circulation rate, and therefore improve bio distribution ofthis nanomedicine.

The present disclosure teaches that by increasing the surface negativecharge of SUVs, via the attachment of anionic entities including, butnot limited to, DNA and RNA, the colloidal stability of these vesiclesis increased. Additionally, the dense spherical arrangement and radialorientation of nucleic acids exhibits unique chemical and biologicalproperties, unlike their linear counterparts. These spherical nucleicacids (SNA) are non-toxic and though anionic, can efficiently entercells without the aid of ancillary cationic transfection agents in anon-immunogenic fashion. These exceptional properties allow their use asdelivery agents for gene regulation in different therapies. Theliposome-template mediated synthesis of SNAs provides an alternativeplatform to metal core SNAs which limits SNAs therapeutic diversity withbioaccumulation of the metal core and inability to encapsulatetherapeutic entities.

Tocopherol modified oligonucleotides and methods of making sucholigonucleotides, liposomal particles and methods of making same, anduses of liposomal particles now will be described more fullyhereinafter. Indeed, the disclosure may be embodied in many differentforms and should not be construed as limited to the embodiments setforth herein. These embodiments are provided in sufficient writtendetail to describe and enable one skilled in the art to make and use theinvention, along with disclosure of the best mode for practicing theinvention, as defined by the claims and equivalents thereof.

Likewise, many modifications and other embodiments of the methodsdescribed herein will come to mind to one of skill in the art to whichthe invention pertains having the benefit of the teachings presented inthe foregoing descriptions and the associated drawings. Therefore, it isto be understood that the invention is not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

Terminology

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of skill in the artto which the invention pertains. Although any methods and materialssimilar to or equivalent to those described herein can be used in thepractice or testing of the invention, the preferred methods andmaterials are described herein.

Certain terms are first defined. Additional terms are defined throughoutthe specification.

For the sake of brevity, a description of an embodiment of thedisclosure in terms of a small unilamellar vesicle (SUV), a liposomalSNA (LSNA), a liposomal particle, or a spherical nucleic acid (SNA) mayalso be applicable to an embodiment that uses any of the other foregoingterms. By way of example, a method of regulating gene expression using aliposomal SNA may also be described herein as a method of regulatinggene expression using a liposomal particle. Small unilamellar vesicles(SUVs) are liposomal particles of sub-100 nanometer size and are used asthe precursors to LSNAs. SUVs and LSNAs, as such, can be consideredsubclasses of liposomal particles.

Terms used herein are intended as “open” terms (for example, the term“including” should be interpreted as “including but not limited to,” theterm “having” should be interpreted as “having at least,” the term“includes” should be interpreted as “includes but is not limited to”).

Furthermore, in those instances where a convention analogous to “atleast one of A, B and C, etc.” is used, in general such a constructionis intended in the sense of one having ordinary skill in the art wouldunderstand the convention (for example, “a system having at least one ofA, B and C” would include but not be limited to systems that have Aalone, B alone, C alone, A and B together, A and C together, B and Ctogether, and/or A, B, and C together). It will be further understood bythose within the art that virtually any disjunctive word and/or phrasepresenting two or more alternative terms, whether in the description orfigures, should be understood to contemplate the possibilities ofincluding one of the terms, either of the terms, or both terms. Forexample, the phrase “A or B” will be understood to include thepossibilities of “A or B” or “A and B.”

All language such as “from,” “to,” “up to,” “at least,” “greater than,”“less than,” and the like include the number recited and refer to rangeswhich can subsequently be broken down into sub-ranges as discussedabove.

A range includes each individual member. Thus, for example, a grouphaving 1-3 members refers to groups having 1, 2, or 3 members.Similarly, a group having 6 members refers to groups having 1, 2, 3, 4,or 6 members, and so forth.

The modal verb “may” refers to the preferred use or selection of one ormore options or choices among the several described embodiments orfeatures contained within the same. Where no options or choices aredisclosed regarding a particular embodiment or feature contained in thesame, the modal verb “may” refers to an affirmative act regarding how tomake or use an aspect of a described embodiment or feature contained inthe same, or a definitive decision to use a specific skill regarding adescribed embodiment or feature contained in the same. In this lattercontext, the modal verb “may” has the same meaning and connotation asthe auxiliary verb “can.”

As used herein, the articles “a” and “an” refer to one or to more thanone (for example, to at least one) of the grammatical object of thearticle.

“About” and “approximately” shall generally mean an acceptable degree oferror for the quantity measured given the nature or precision of themeasurements. Exemplary degrees of error are within 20-25 percent (%),typically, within 10%, and more typically, within 5% of a given value orrange of values.

The chemical structures described herein are named according to IUPACnomenclature rules and include art-accepted common names andabbreviations where appropriate. The IUPAC nomenclature can be derivedwith chemical structure drawing software programs, such as ChemDraw®(PerkinElmer, Inc.), ChemDoodle® (iChemLabs, LLC) and Marvin (ChemAxonLtd.). The chemical structure controls in the disclosure to the extentthat an IUPAC name is misnamed or otherwise conflicts with the chemicalstructure disclosed herein.

Headings, for example, (A), (B), (i) etc., are presented merely for easeof reading the specification and claims. The use of headings in thespecification or claims does not require the steps or elements beperformed in alphabetical or numerical order or the order in which theyare presented.

The present disclosure describes novel particles, termed liposomalparticles, methods of making the same, and uses of these particles. Thepresent liposomal particles are advantageous over other known liposomalbased materials in that they are stable at a particle size that issmaller than other known liposomal particles, and the dense layer of DNAincreases particle stability in a body, and therefore increases thecirculation rate of liposomal vesicles, which improves bio-distributionof these particles inside the body.

A. Tocopherol Modified Oligonucleotides

In a first embodiment, an architecture comprising a tocopherol modifiedoligonucleotide is disclosed. A tocopherol-modified oligonucleotidecomprises a lipophilic end and a non-lipophilic end. The lipophilic endcomprises tocopherol, and may be chosen from the group consisting of atocopherol derivative, alpha-tocopherol, beta-tocopherol,gamma-tocopherol and delta-tocopherol. The lipophilic end, in furtherembodiments, comprises palmitoyl, dipalmitoyl, stearyl, or distearyl.

The non-lipophilic end of the tocopherol-modified oligonucleotide is anoligonucleotide. The oligonucleotide is either RNA or DNA. The RNA canbe an inhibitory RNA (RNAi) that performs a regulatory function, and ischosen from the group consisting of a small RNAi that is selected fromthe group consisting of a small inhibitory RNA (siRNA), an RNA thatforms a triplex with double stranded DNA, and a ribozyme. Alternatively,the RNA is microRNA that performs a regulatory function. In stillfurther embodiments, the RNA is a piwi-interacting RNA (piRNA). The DNAis, in some embodiments, an antisense-DNA.

Oligonucleotides contemplated for use according to the disclosure arefrom about 5 to about 100 nucleotides in length. Methods andcompositions are also contemplated wherein the oligonucleotide is about5 to about 90 nucleotides in length, about 5 to about 80 nucleotides inlength, about 5 to about 70 nucleotides in length, about 5 to about 60nucleotides in length, about 5 to about 50 nucleotides in length about 5to about 45 nucleotides in length, about 5 to about 40 nucleotides inlength, about 5 to about 35 nucleotides in length, about 5 to about 30nucleotides in length, about 5 to about 25 nucleotides in length, about5 to about 20 nucleotides in length, about 5 to about 15 nucleotides inlength, about 5 to about 10 nucleotides in length, and alloligonucleotides intermediate in length of the sizes specificallydisclosed to the extent that the oligonucleotide is able to achieve thedesired result. Accordingly, oligonucleotides of 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, and 100nucleotides in length are contemplated.

Modified Oligonucleotides

Specific examples of oligonucleotides include those containing modifiedbackbones or non-natural internucleoside linkages. Oligonucleotideshaving modified backbones include those that retain a phosphorus atom inthe backbone and those that do not have a phosphorus atom in thebackbone. Modified oligonucleotides that do not have a phosphorus atomin their internucleoside backbone are considered to be within themeaning of “oligonucleotide.”

Modified oligonucleotide backbones containing a phosphorus atom include,for example, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonates,5′-alkylene phosphonates and chiral phosphonates, phosphinates,phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphatesand boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogsof these, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Also contemplated are oligonucleotides having inverted polaritycomprising a single 3′ to 3′ linkage at the 3′-most internucleotidelinkage, i.e. a single inverted nucleoside residue which may be abasic(the nucleotide is missing or has a hydroxyl group in place thereof).Salts, mixed salts and free acid forms are also contemplated.Representative United States patents that teach the preparation of theabove phosphorus-containing linkages include, U.S. Pat. Nos. 3,687,808;4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423;5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939;5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821;5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599;5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050, thedisclosures of which are incorporated by reference herein.

Modified oligonucleotide backbones that do not include a phosphorus atomtherein have backbones that are formed by short chain alkyl orcycloalkyl internucleoside linkages, mixed heteroatom and alkyl orcycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages; siloxane backbones; sulfide, sulfoxideand sulfone backbones; formacetyl and thioformacetyl backbones;methylene formacetyl and thioformacetyl backbones; riboacetyl backbones;alkene containing backbones; sulfamate backbones; methyleneimino andmethylenehydrazino backbones; sulfonate and sulfonamide backbones; amidebackbones; and others having mixed N, O, S and CH₂ component parts. See,for example, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134;5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257;5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086;5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704;5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and5,677,439, the disclosures of which are incorporated herein by referencein their entireties.

In still other embodiments, oligonucleotide mimetics wherein both one ormore sugar and/or one or more internucleotide linkage of the nucleotideunits are replaced with “non-naturally occurring” groups. In one aspect,this embodiment contemplates a peptide nucleic acid (PNA). In PNAcompounds, the sugar-backbone of an oligonucleotide is replaced with anamide containing backbone. See, for example U.S. Pat. Nos. 5,539,082;5,714,331; and 5,719,262, and Nielsen et al., 1991, Science, 254:1497-1500, the disclosures of which are herein incorporated byreference.

In still other embodiments, oligonucleotides are provided withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and including —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂—,—CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂—described in U.S. Pat. Nos. 5,489,677, and 5,602,240. Also contemplatedare oligonucleotides with morpholino backbone structures described inU.S. Pat. No. 5,034,506.

In various forms, the linkage between two successive monomers in theoligonucleotide consists of 2 to 4, desirably 3, groups/atoms selectedfrom —CH₂—, —O—, —S—, —NR^(H)—, >C═O, >C═NR^(H), >C═S, —Si(R″)₂—, —SO—,—S(O)₂—, —P(O)₂—, —PO(BH₃)—, —P(O,S)—, —P(S)₂—, —PO(R″)—, —PO(OCH₃)—,and —PO(NHR^(H))—, where RH is selected from hydrogen and C₁₋₄-alkyl,and R″ is selected from C₁₋₆-alkyl and phenyl. Illustrative examples ofsuch linkages are —CH₂—CH₂—CH₂—, —CH₂—CO—CH₂—, —CH₂—CHOH—CH₂—,—O—CH₂—O—, —O—CH₂—CH₂—, —O—CH₂—CH═ (including R⁵ when used as a linkageto a succeeding monomer), —CH₂—CH₂—O—, —NR^(H)CH₂—CH₂—, —CH₂CH₂—NR^(H)—,—CH₂—NR^(H)—CH₂—, —O—CH₂—CH₂—NR^(H)—, —NR^(H)—CO—O—, —NR^(H)—CO—NR^(H)—,—NR^(H)—CS—NR^(H)—, —NR^(H)—C(═NR^(H))—NR^(H)—,—NR^(H)—CO—CH₂—NR^(H)—O—CO—O—, —O—CO—CH₂—O—, —O—CH₂—CO—O—,—CH₂—CO—NR^(H)—, —O—CO—NR^(H)—, —NR^(H)—CO—CH₂—, —O—CH₂—CO—NR^(H)—,—O—CH₂—CH₂—NR^(H)—, —CH═N—O—, —CH₂—NR^(H)—O—, —CH₂—O—N═ (including R⁵when used as a linkage to a succeeding monomer), —CH₂—O—NR^(H)—,—CO—NR^(H)—CH₂—, —CH₂—NR^(H)—O—, CH₂—NR^(H)—CO—, —O—NR^(H)—CH₂—,—O—NR^(H)—, —O—CH₂—S—, —S—CH₂—O—, —CH₂—CH₂—S—, —O—CH₂—CH₂—S—, —S—CH₂—CH═(including R⁵ when used as a linkage to a succeeding monomer),—S—CH₂—CH₂—, —S—CH₂—CH₂—O—, —S—CH₂—CH₂—S—, —CH₂—S—CH₂—, —CH₂—SO—CH₂—,—CH₂—SO₂—CH₂—, —O—SO—O—, —O—S(O)₂—O—, —O—S(O)₂—CH₂—, —O—S(O)₂—NR^(H)—,—NR^(H)—S(O)₂—CH₂—; —O—S(O)₂—CH₂—, —O—P(O)₂—O—, —O—P(O,S)—O—,—O—P(S)₂—O—, —S—P(O)₂—O—, —S—P(O,S)—O—, —S—P(S)₂—O—, —O—P(O)₂—S—,—O—P(O,S)—S—, —O—P(S)₂—S—, —S—P(O)₂—S—, —S—P(O,S)—S—, —S—P(S)₂—S—,—O—PO(R″)—O—, —O—PO(OCH₃)—O—, —O—PO(OCH₂CH₃)—O—, —O—PO(O CH₂CH₂S—R)—O—,—O—PO(BH₃)—O—, —O—PO(NHR^(N))—O—, —O—P(O)₂—NR^(H) H—, —NR^(H)—P(O)₂—O—,—O—P(O,NR^(H))—O—, —CH₂—P(O)₂—O—, —O—P(O)₂—CH₂—, and —O—Si(R″)₂—O—;among which —CH₂—CO—NR^(H)—, —CH₂—NR^(H)—O—, —S—CH₂—O—,—O—P(O)₂—O—O—P(—O,S)—O—, —O—P(S)₂—O—, —NR^(H) P(O)₂—O—,—O—P(O,NR^(H))—O—, —O—PO(R″)—O—, —O—PO(CH₃)—O—, and —O—PO(NHR^(N))—O—,where RH is selected form hydrogen and C₁₋₄-alkyl, and R″ is selectedfrom C₁₋₆-alkyl and phenyl, are contemplated. Further illustrativeexamples are given in Mesmaeker et. al., 1995, Current Opinion inStructural Biology, 5: 343-355 and Susan M. Freier and Karl-HeinzAltmann, 1997, Nucleic Acids Research, vol 25: pp 4429-4443.

Still other modified forms of oligonucleotides are described in detailin U.S. Patent Application No. 20040219565, the disclosure of which isincorporated by reference herein in its entirety.

Modified oligonucleotides may also contain one or more substituted sugarmoieties. In certain aspects, oligonucleotides comprise one of thefollowing at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, orN-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Other embodiments includeO[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃,O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, where n and m are from1 to about 10. Other oligonucleotides comprise one of the following atthe 2′ position: C₁ to C₁₀ lower alkyl, substituted lower alkyl,alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃,OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of anoligonucleotide, or a group for improving the pharmacodynamic propertiesof an oligonucleotide, and other substituents having similar properties.In one aspect, a modification includes 2′-methoxyethoxy(2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martinet al., 1995, Helv. Chim. Acta, 78: 486-504) i.e., an alkoxyalkoxygroup. Other modifications include 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂₀N(CH₃)₂ group, also known as 2′-DMAOE, as described in examplesherein below, and 2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₃)₂, also described in examples herein below.

Still other modifications include 2′-methoxy (2′-O—CH₃), 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂—CH═CH₂),2′-O-allyl(2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modificationmay be in the arabino (up) position or ribo (down) position. In oneaspect, a 2′-arabino modification is 2′-F. Similar modifications mayalso be made at other positions on the oligonucleotide, for example, atthe 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′linked oligonucleotides and the 5′ position of 5′ terminal nucleotide.Oligonucleotides may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar. See, for example, U.S.Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265;5,658,873; 5,670,633; 5,792,747; and 5,700,920, the disclosures of whichare incorporated herein by reference in their entireties.

In one aspect, a modification of the sugar includes Locked Nucleic Acids(LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′ carbonatom of the sugar ring, thereby forming a bicyclic sugar moiety. Thelinkage is in certain aspects is a methylene (—CH₂—)_(n) group bridgingthe 2′ oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs andpreparation thereof are described in WO 98/39352 and WO 99/14226.

Oligonucleotides may also include base modifications or substitutions.As used herein, “unmodified” or “natural” bases include the purine basesadenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). Modified bases include other synthetic andnatural bases such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and otheralkyl derivatives of adenine and guanine, 2-propyl and other alkylderivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil andcytosine and other alkynyl derivatives of pyrimidine bases, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil,8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other8-substituted adenines and guanines, 5-halo particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine,8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and3-deazaguanine and 3-deazaadenine. Further modified bases includetricyclic pyrimidines such as phenoxazinecytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzox-azin-2(3H)-one),carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindolecytidine (H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modifiedbases may also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further bases includethose disclosed in U.S. Pat. No. 3,687,808, those disclosed in TheConcise Encyclopedia Of Polymer Science And Engineering, pages 858-859,Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed byEnglisch et al., 1991, Angewandte Chemie, International Edition, 30:613, and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, pages 289-302, Crooke, S. T. and Lebleu, B.,ed., CRC Press, 1993. Certain of these bases are useful for increasingthe binding affinity and include 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. and are, in certain aspects combinedwith 2′-O-methoxyethyl sugar modifications. See, U.S. Pat. Nos.3,687,808, 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066;5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985;5,830,653; 5,763,588; 6,005,096; 5,750,692 and 5,681,941, thedisclosures of which are incorporated herein by reference.

A “modified base” or other similar term refers to a composition whichcan pair with a natural base (e.g., adenine, guanine, cytosine, uracil,and/or thymine) and/or can pair with a non-naturally occurring base. Incertain aspects, the modified base provides a T_(m) differential of 15,12, 10, 8, 6, 4, or 2° C. or less. Exemplary modified bases aredescribed in EP 1 072 679 and WO 97/12896.

By “nucleobase” is meant the naturally occurring nucleobases adenine(A), guanine (G), cytosine (C), thymine (T) and uracil (U) as well asnon-naturally occurring nucleobases such as xanthine, diaminopurine,8-oxo-N⁶-methyladenine, 7-deazaxanthine, 7-deazaguanine,N⁴,N⁴-ethanocytosin, N′,N′-ethano-2,6-diaminopurine, 5-methylcytosine(mC), 5-(C³—C⁶)-alkynyl-cytosine, 5-fluorouracil, 5-bromouracil,pseudoisocytosine, 2-hydroxy-5-methyl-4-tr-iazolopyridin, isocytosine,isoguanine, inosine and the “non-naturally occurring” nucleobasesdescribed in Benner et al., U.S. Pat. No. 5,432,272 and Susan M. Freierand Karl-Heinz Altmann, 1997, Nucleic Acids Research, vol. 25: pp4429-4443. The term “nucleobase” thus includes not only the known purineand pyrimidine heterocycles, but also heterocyclic analogues andtautomers thereof. Further naturally and non-naturally occurringnucleobases include those disclosed in U.S. Pat. No. 3,687,808 (Merigan,et al.), in Chapter 15 by Sanghvi, in Antisense Research andApplication, Ed. S. T. Crooke and B. Lebleu, CRC Press, 1993, inEnglisch et al., 1991, Angewandte Chemie, International Edition, 30:613-722 (see especially pages 622 and 623, and in the ConciseEncyclopedia of Polymer Science and Engineering, J. I. Kroschwitz Ed.,John Wiley & Sons, 1990, pages 858-859, Cook, Anti-Cancer Drug Design1991, 6, 585-607, each of which are hereby incorporated by reference intheir entirety). The term “nucleosidic base” or “base unit” is furtherintended to include compounds such as heterocyclic compounds that canserve like nucleobases including certain “universal bases” that are notnucleosidic bases in the most classical sense but serve as nucleosidicbases. Especially mentioned as universal bases are 3-nitropyrrole,optionally substituted indoles (e.g., 5-nitroindole), and optionallysubstituted hypoxanthine. Other desirable universal bases include,pyrrole, diazole or triazole derivatives, including those universalbases known in the art.

B. Methods of Making Tocopherol Modified Oligonucleotides

In a second embodiment, methods of making tocopherol oligonucleotidesare disclosed. First, an oligonucleotide andphosphoramidite-modified-tocopherol are provided. Then, theoligonucleotide is exposed to the phosphoramidite-modified-tocopherol tocreate the tocopherol modified oligonucleotide. While not meant to belimiting, any chemistry to one of skill in the art can be used to attachthe tocopherol to the oligonucleotide, including amide linking or clickchemistry.

C. Liposomal Particles

In a third embodiment, liposomal particles are disclosed. The liposomalparticle has at least a substantially spherical geometry, an internalside and an external side, and comprises a lipid bilayer. The lipidbilayer is comprised of a first-lipid and a second-lipid. Thefirst-lipid and second-lipid are, in some embodiments, the same. Infurther embodiments, the first-lipid and second-lipid are different.

The first-lipid is chosen from the phosphocholine family of lipids orthe phosphoethanolamine family of lipids. While not meant to belimiting, the first-lipid is chosen from group consisting of1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1,2-dimyristoyl-sn-phosphatidylcholine (DMPC),1-palmitoyl-2-oleoyl-sn-phosphatidylcholine (POPC),1,2-distearoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DSPG),1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DOPG),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), and1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DPPE).

The second-lipid is chosen from the phosphocholine family of lipids orthe phosphoethanolamine family of lipids. While not meant to belimiting, the second-lipid is chosen from group consisting of1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1,2-dimyristoyl-sn-phosphatidylcholine (DMPC),1-palmitoyl-2-oleoyl-sn-phosphatidylcholine (POPC),1,2-distearoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DSPG),1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DOPG),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), and1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DPPE).

The liposomal particle further comprises a tocopherol modifiedoligonucleotide wherein the lipophilic end of the tocopherol modifiedoligonucleotide is absorbed into the lipid bilayer. The tocopherol ischosen from the group consisting of alpha-tocopherol, beta-tocopherol,gamma-tocopherol and delta-tocopherol. The non-lipophilic end of thetocopherol modified oligonucleotide is an oligonucleotide. Thisoligonucleotide is, in various embodiments, either RNA or DNA. The RNAcan be an inhibitory RNA (RNAi) that performs a regulatory function, andin various embodiments is selected from the group consisting of a smallinhibitory RNA (siRNA), an RNA that forms a triplex with double strandedDNA, and a ribozyme. Alternatively, and in further embodiments, the RNAis microRNA that performs a regulatory function. The DNA is optionallyan antisense-DNA. In still further embodiments, the RNA is apiwi-interacting RNA (piRNA).

Put another way, the disclosure provides a liposomal particle, saidliposomal particle having a substantially spherical geometry, saidliposomal particle comprising a lipid bilayer comprising a plurality oflipid groups; and an oligonucleotide. In various embodiments, theoligonucleotide is a modified oligonucleotide. In some embodiments, theplurality of lipid groups comprises a lipid selected from the groupconsisting of the phosphatidylcholine, phosphatidylglycerol, andphosphatidylethanolamine family of lipids. The oligonucleotide, infurther embodiments is an oligonucleotide-lipid conjugate containing alipophilic tethered group, wherein said lipophilic tethered group isadsorbed into the lipid bilayer. The lipophilic tethered groupcomprises, in various embodiments, tocopherol, palmitoyl, dipalmitoyl,stearyl, distearyl, or cholesterol.

Alternatively, the liposomal particle further comprises a therapeuticagent encapsulated on the internal side of the liposomal particle. Infurther embodiments, a liposomal particle of the disclosure furthercomprises a therapeutic agent that is either directly or indirectlyattached to the liposomal particle. Indirect attachment includes, forexample and without limitation, attachment to an oligonucleotide that isin turn attached to the liposomal particle.

In some embodiments, the liposomal particle further comprises adiagnostic agent encapsulated on the internal side of the liposomalparticle. This diagnostic agent is in some embodiments gadolinium.

With respect to the surface density of oligonucleotides on the surfaceof a liposomal particle of the disclosure, it is contemplated that aliposomal particle as described herein comprises from about 1 to about100 oligonucleotides on its surface. In various embodiments, a liposomalparticle comprises from about 10 to about 100, or from 10 to about 90,or from about 10 to about 80, or from about 10 to about 70, or fromabout 10 to about 60, or from about 10 to about 50, or from about 10 toabout 40, or from about 10 to about 30, or from about 10 to about 20oligonucleotides on its surface. In further embodiments, a liposomalparticle comprises at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90,or 100 oligonucleotides on its surface.

D. Methods of Making Liposomal Particles

In a fourth embodiment, methods of making liposomal particles aredisclosed. First, a phospholipid, solvent, and a tocopherol modifiedoligonucleotide are provided. Then, the phospholipid is added to thesolvent to form a first mixture comprising liposomes. The size of theliposomes in the first mixture is between about 100 nanometers and about150 nanometers.

Next, the liposomes are disrupted to create a second mixture comprisingliposomes and small unilamellar vesicles (SUV). The size of theliposomes and SUVs in the second mixture is between about 20 nanometersand about 150 nanometers.

Next, the SUVs having a particle size between about 20 nanometers andabout 50 nanometers are isolated from the second mixture. Finally, thetocopherol modified oligonucleotide is added to the isolated SUVs tomake a liposomal particle.

The particle size of the liposomal particles created by a method of thedisclosure is less than or equal to about 50 nanometers. In someembodiments, a plurality of liposomal particles is produced and theparticles in the plurality have a mean diameter of less than or equal toabout 50 nanometers (e.g., about 5 nanometers to about 50 nanometers, orabout 5 nanometers to about 40 nanometers, or about 5 nanometers toabout 30 nanometers, or about 5 nanometers to about 20 nanometers, orabout 10 nanometers to about 50 nanometers, or about 10 nanometers toabout 40 nanometers, or about 10 nanometers to about 30 nanometers, orabout 10 nanometers to about 20 nanometers). In further embodiments, theparticles in the plurality of liposomal particles created by a method ofthe disclosure have a mean diameter of less than or equal to about 20nanometers, or less than or equal to about 25 nanometers, or less thanor equal to about 30 nanometers, or less than or equal to about 35nanometers, or less than or equal to about 40 nanometers, or less thanor equal to about 45 nanometers.

Put another way, in some aspects the disclosure provides a method ofmaking a liposomal particle, comprising adding a phospholipid to asolvent to form a first mixture, said first mixture comprising aplurality of liposomes; disrupting said plurality of liposomes to createa second mixture, said second mixture comprising a liposome and a smallunilamellar vesicle (SUV); isolating said SUV from said second mixture,said SUV having a particle size between about 20 nanometers and 50nanometers; and adding an oligonucleotide to the isolated SUV to makethe liposomal particle.

E. Uses of Liposomal Particles in Gene Regulation/Therapy

Methods for inhibiting gene product expression provided herein includethose wherein expression of the target gene product is inhibited by atleast about 5%, at least about 10%, at least about 15%, at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about55%, at least about 60%, at least about 65%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, at least about 99%, or 100% compared to gene productexpression in the absence of a liposome SNA. In other words, methodsprovided embrace those which results in essentially any degree ofinhibition of expression of a target gene product.

The degree of inhibition is determined in vivo from a body fluid sampleor from a biopsy sample or by imaging techniques well known in the art.Alternatively, the degree of inhibition is determined in a cell cultureassay, generally as a predictable measure of a degree of inhibition thatcan be expected in vivo resulting from use of a specific type ofliposomal SNA and a specific oligonucleotide.

In some aspects of the disclosure, it is contemplated that a liposomalparticle performs both a gene inhibitory function as well as atherapeutic agent delivery function. In such aspects, a therapeuticagent is encapsulated in a liposomal particle of the disclosure and theparticle is additionally functionalized with one or moreoligonucleotides designed to effect inhibition of target geneexpression. In further embodiments, a therapeutic agent is attached to aliposomal particle of the disclosure.

In various aspects, the methods include use of an oligonucleotide whichis 100% complementary to the target polynucleotide, i.e., a perfectmatch, while in other aspects, the oligonucleotide is at least (meaninggreater than or equal to) about 95% complementary to the polynucleotideover the length of the oligonucleotide, at least about 90%, at leastabout 85%, at least about 80%, at least about 75%, at least about 70%,at least about 65%, at least about 60%, at least about 55%, at leastabout 50%, at least about 45%, at least about 40%, at least about 35%,at least about 30%, at least about 25%, at least about 20% complementaryto the polynucleotide over the length of the oligonucleotide to theextent that the oligonucleotide is able to achieve the desired degree ofinhibition of a target gene product.

It is understood in the art that the sequence of an antisense compoundneed not be 100% complementary to that of its target nucleic acid to bespecifically hybridizable. Moreover, an oligonucleotide may hybridizeover one or more segments such that intervening or adjacent segments arenot involved in the hybridization event (e.g., a loop structure orhairpin structure). The percent complementarity is determined over thelength of the oligonucleotide. For example, given an antisense compoundin which 18 of 20 nucleotides of the antisense compound arecomplementary to a 20 nucleotide region in a target polynucleotide of100 nucleotides total length, the oligonucleotide would be 90 percentcomplementary. In this example, the remaining noncomplementarynucleotides may be clustered or interspersed with complementarynucleobases and need not be contiguous to each other or to complementarynucleotides. Percent complementarity of an antisense compound with aregion of a target nucleic acid can be determined routinely using BLASTprograms (basic local alignment search tools) and PowerBLAST programsknown in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403-410;Zhang and Madden, Genome Res., 1997, 7, 649-656).

Accordingly, in a fifth embodiment, methods of utilizing liposomalparticles in gene regulation therapy are provided. This method comprisesthe step of hybridizing a polynucleotide encoding said gene product withone or more oligonucleotides complementary to all or a portion of saidpolynucleotide, said oligonucleotide being attached to a liposomalparticle, wherein hybridizing between said polynucleotide and saidoligonucleotide occurs over a length of said polynucleotide with adegree of complementarity sufficient to inhibit expression of said geneproduct. The liposomal particle has a diameter that is about less thanor equal to 50 nanometers. The inhibition of gene expression may occurin vivo or in vitro.

The oligonucleotide utilized in this method is either RNA or DNA. TheRNA can be an inhibitory RNA (RNAi) that performs a regulatory function,and in various embodiments is selected from the group consisting of asmall inhibitory RNA (siRNA), an RNA that forms a triplex with doublestranded DNA, and a ribozyme. Alternatively, the RNA is microRNA thatperforms a regulatory function. The DNA is, in some embodiments, anantisense-DNA.

In another aspect of the disclosure, a liposomal particle is used in amethod for treating a traumatic brain injury (TBI). In the UnitedStates, there have been over 244,000 cases of TBI in the military since2000, and it is the leading cause of death and disability in peopleunder the age of 45. Further, it is currently difficult to predict theneurological outcome of “mild severity” incidents, and the secondaryphase of the injury (e.g., inflammation, ischemia, and apoptosis) isvery difficult to treat.

Thus, in some embodiments, methods of the disclosure are directed to theuse of a liposomal particle designed to target and regulate theexpression of a gene product implicated in TBI. For example and withoutlimitation, the target gene product is selected from the groupconsisting of histone deacetylase (HDAC), BCL2-associated X (BAX), amatrix metallopeptidase/metalloproteinase (MMP; including, withoutlimitation, matrix metallopeptidase 9 (MMP-9)), a hypoxia-induciblefactor (HIF; including, without limitation, hypoxia inducible factor 1alpha (HIF1-α)), and calpain.

F. Use of Liposomal Particles in Immune Regulation

Toll-like receptors (TLRs) are a class of proteins, expressed insentinel cells, that plays a key role in regulation of innate immunesystem. The mammalian immune system uses two general strategies tocombat infectious diseases. Pathogen exposure rapidly triggers an innateimmune response that is characterized by the production ofimmunostimulatory cytokines, chemokines and polyreactive IgM antibodies.The innate immune system is activated by exposure to Pathogen AssociatedMolecular Patterns (PAMPs) that are expressed by a diverse group ofinfectious microorganisms. The recognition of PAMPs is mediated bymembers of the Toll-like family of receptors. TLR receptors, such as TLR4, TLR 8 and TLR 9 that response to specific oligonucleotide are locatedinside special intracellular compartments, called endosomes. Themechanism of modulation of TLR 4, TLR 8 and TLR9 receptors is based onDNA-protein interactions.

Synthetic immunostimulatory oligonucleotides that contain CpG motifsthat are similar to those found in bacterial DNA stimulate a similarresponse of the TLR receptors. Therefore immunomodulatory ODNs havevarious potential therapeutic uses, including treatment of immunedeficiency and cancer. Employment of liposomal nanoparticlesfunctionalized with immunomodulatory ODNs will allow for increasedpreferential uptake and therefore increased therapeutic efficacy.Notably, smaller particles (25 to 40 nm) such as those provided hereinpenetrate tissue barriers more efficiently, therefore providing moreeffective activation of innate immune responses. Thus, small liposomalnanoparticles of 30 nm in size, functionalized with stabilized withfunctional CpG motif-containing DNA, would provide enhanced therapeuticeffect.

Down regulation of the immune system would involve knocking down thegene responsible for the expression of the Toll-like receptor. Thisantisense approach involves use of liposomal nanoparticlesfunctionalized with specific antisense oligonucleotide sequences toknock out the expression of any toll-like protein.

Accordingly, in a sixth embodiment, methods of utilizing liposomalparticles for modulating toll-like receptors are disclosed. The methodeither up-regulates or down-regulates the Toll-like-receptor through theuse of a TLR agonist or a TLR antagonist, respectively. The methodcomprises contacting a cell having a toll-like receptor with a liposomalparticle. The toll-like receptors modulated include toll-like receptor1, toll-like receptor 2, toll-like receptor 3, toll-like receptor 4,toll-like receptor 5, toll-like receptor 6, toll-like receptor 7,toll-like receptor 8, toll-like receptor 9, toll-like receptor 10,toll-like receptor 11, toll-like receptor 12, and toll-like receptor 13.

G. Use of Liposomal Particles in Nanoflare Technology

In additional aspects of the disclosure, a liposomal particle is used todetect an intracellular target. Such methods are disclosed in U.S. Pat.No. 8,507,200, which is incorporated by reference herein in itsentirety.

Briefly, an oligonucleotide containing a recognition sequence that isspecific for a target molecule is attached to a liposomal particle asdescribed herein. Thus, “recognition sequence” as used herein isunderstood to mean a sequence that is partially or completelycomplementary to a target molecule of interest.

The liposomal particle with attached oligonucleotide containing arecognition sequence is initially associated with a reporter sequence.As used herein, a “reporter sequence” is understood to mean a sequencethat is partially or completely complementary and therefore able tohybridize to the recognition sequence. The reporter sequence is labeledwith a detectable label (such as, without limitation, a fluorophore),and is also referred to as a nanoflare. The reporter sequence is invarious aspects comprised of fewer, the same or more bases than therecognition sequence, such that binding of the recognition sequence toits target molecule causes release of the hybridized reporter sequence,thereby resulting in a detectable and measurable change in the labelattached to the reporter sequence.

The invention is illustrated by the following examples, which are notintended to be limiting in any way.

EXAMPLES Example 1—General

All reagents were obtained from the suppliers in highest purity and usedwithout any further purification. HPLC was performed on a Varian Prostarsystem. UV/Vis was recorded on a Varian Cary 300 spectrophotometer.Fluorescence spectra were obtained on a SPEX FluoroLog fluorometer.

Example 2—Synthesis of Oligonucleotides

Oligonucleotides were synthesized in 1.0 micromolar scale on anautomated DNA synthesizer (ABI 3400, Applied Biosystems, Inc.). Aftercleavage and deprotection with aqueous ammonium hydroxide (55° C., 14h), the DNA was purified by reverse-phase HPLC and quantified by UVspectrometer.

Example 3—Synthesis of Liposomal Particles

The lipid monomer (40 μmol of 1,2-dioleoyl-sn-glycero-3-phosphocholine(DOPC) dissolved in chloroform) was added to a 20 mL vial and thenevaporated before overnight lyophilization to remove the solventresulting in a thin lipid film. The film was then rehydrated with HBSbuffer (5.0 mL, 20 mM Hepes buffer, 150 mM NaCl at pH 7.4) followed byvigorous mixing to form a liposomal suspension and was thenprobe-sonicated in an ice bath for 30 min without pulsating. Theresulting suspension was then ultracentrifuged at 104,986 g and 4° C.for 90 min. The phospholipid concentration was calculated usingelemental analysis.

Next, the DNA/RNA strands were synthesized with the α-tocopherolmodification via standard solid-phase phosphoramidite chemistry on anExpedite Nucleotide Synthesis System. The strands were cleaved from thesolid support and purified by reverse-phase high performance liquidchromatography.

Lastly, the appropriate DNA/RNA (16 μM) was added to the 1.3 mM solutionof SUVs and allowed to stir overnight. The particles were then purifiedthe next day by centrifugation filters with cut-off of 100 kDa. Theparticles were then analyzed via TEM and dynamic light scattering. Gelelectrophoresis of liposomal particles encapsulated with FITC andsurface functionalized with CY5-labeled DNA is shown in FIG. 13 .

Example 4—Visualization of the Cellular Uptake of Liposomal Particles

To visualize the cellular uptake of LSNAs, HeLa cells were grown on aLab-Tek® II Chamber #1.5 German Coverglass System (Nalge NuncInternational) overnight and incubated with Cy5-labled LSNAs (0.1 μM ofDNA concentration). After 16 hours of incubation, the media was replacedwith fresh media, and live cells were stained with Hoechst 33342(Invitrogen) following the manufacturer's instructions. All images wereobtained with a Zeiss 510 LSM at 40× magnification using a Mai Tai 3308laser (Spectra-Physics). Fluorescence emission was collected at 390-465nm and 650-710 nm, with excitation at 729 and 633 nm respectively (FIG.5 ). The left panel of FIG. 5 shows entry of liposomal fluorescein intoHeLa cells, while the right panel of FIG. 5 shows co-localization offluorescein and Cy5 suggesting delivery of the entire liposome into thecell.

Example 4—Cell Viability

The cytotoxicity of liposomal particles was evaluated with a AlamarBlue® Assay (Invitrogen). Briefly, HeLa cells were seeded on a 96 wellplate in 200 μL of media and incubated for 24 hours. The cells were thentreated with FITC encapsulated bare SUVs and DNA functionalized LSNAs atvarying concentrations of phospholipid concentrations (0, 32.5, 65,162.5 μM). After 16 hours, medium was removed, cells were washed withPBS 3 times and then incubated with 90 μL fresh culture medium inaddition to 10 μL of alamar blue reagent for 4 hours. They were thenanalyzed by checking the excitation at 560 nm and emission at 590 nm.

Example 5—Preparation of SUVs Materials

The 1,2-dioleoyl-sn-glycero-3-phosphocholine lipid monomer (DOPC), werepurchased from Avanti Polar Lipids, Inc. either in dry powder form or ina chloroform solution and used without further purification.Phosphoramidites and other DNA synthesis reagents were purchased fromGlen Research, Inc., at the highest purity and were used as receivedfrom the manufacturer.

Instrumentation

Lyophilization was carried out using a Freezone Lyohilizer (Labconco,Kansas City, MO). Sonication was conducted using a titanium-alloy solidprobe sonicator (500 watt Vibra-Cell™ VC 505, Sonics & Materials, Inc.,Newtown, CT) set at 40% intensity of 20 kHz without pulsing.Ultracentrifugation was carried out using Beckman-Coulter Avanti J-30I(Beckmann-Coulter, Inc., Indianapolis, IN). Transmission electronmicroscopy (TEM) was performed using Hitachi-2300 STEM electronmicroscope. Dynamic light scattering (DLS) was collected using a MalvernZetasizer Nano-ZS (Malvern Instruments, UK). MALDI-ToF analysis wasperformed using Bruker Autoflex III SmartBean mass spectrometer (BrukerDaltonics Inc., MA, USA). Fluorescence measurements were carried out onFluorlog-3 system (HORIBA Jobin Yvon Inc., NJ, USA). UV-Vis spectroscopywas collected using Cary 5000 UV-Vis spectrophotometer. (Varian Inc.,CA, USA).

Oligonucleotide Synthesis

The oligonucleotides were synthesized using automated solid-supportphosphoramidite synthesis on an Expedite 8909 Nucleotide SynthesisSystem (MM48 Synthesizer, Bioautomation) using DCI as an activator.Tocopherol phosphoramidite was coupled via an automated protocol usingextended 15 minutes coupling time. After the completion of solid phasesynthesis, the oligonucleotide strands were cleaved from the solidsupport using an overnight treatment with aqueous ammonium hydroxide(28-30% aqueous solution, Aldrich), after which time the excess of theammonia was removed using a gentle flow of nitrogen gas (house nitrogenwas used). The oligonucleotides were purified using Microsorb C18 columnon a reverse-phase high pressure liquid chromatography (HPLC, Varian)using a gradient of TEAA (triethylammonium acetate) buffer andacetonitrile (gradient: 10% v/v to 100% v/v acetonitrile over 30 min).The collected fractions containing product were concentrated on alyophilizer. The obtained oligonucleotides were re-suspended in nanopurewater and purity was analyzed using MALDI-TOF and denaturing acrylamidegel electrophoresis techniques.

TABLE 1 Oligonucleotide sequences used in the experiments.Name of the strand Application Sequence (5′-3′) Cy5 labeled T₂₅Size analysis, 5′-Cy5-T₂₅-tocopherol-3′ strand DNA density(SEQ ID NO: 1) determination and stability studies Melt strand 1Melt analysis 5′-tocopherol-A₁₀-TCT CTT GGA-3′ (SEQ ID NO: 2)Melt strand 2 Melt analysis 5′-TGC GTA GAC-A₁₀ tocopherol-3′(SEQ ID NO: 3) Linker strand Melt analysis 5′-ACG CAT CTG TCC AAG AGA-3′(SEQ ID NO: 4) HER2 antisense Gene regulation5′-CTC CAT GGT GCT CAC-T₁₀- tocopherol-3′ (SEQ ID NO: 5)Cy5 labeled HER2 Imaging and Cellular 5′-Cy5-CTC CAT GGT GCT CAC-T₁₀-antisense uptake tocopherol-3′ (SEQ ID NO: 6) Scrambled antisenseGene regulation 5′-GAG CTG CAC GCT GCC GTC A-T₁₀- tocopherol-3′(SEQ ID NO: 7)

Synthesis of Small Unilamellar Vesicles

The volume of lipid monomer stock solution (25-50 mg) was added to a 20mL vial and placed into a 25 mL glass vial and the solvent was carefullyevaporated using a stream of nitrogen. The obtained lipid monomer wasfurther dried overnight under vacuum to remove the residual chloroform.The resulting lipid film was then hydrated with 20 mM HBS (5.0 mL)followed by vortexing the vial to form a liposomal suspension. Thissuspension was further probe-sonicated for 30 min keeping thetemperature of the lipid mixture below 10° C. (cooling with an ice-waterbath). After the sonication, the suspension was subjected toultracentrifugation at 100,000×g for 90 min at 12° C. After thecentrifugation, the clear supernatant containing the desired smallunilamellar vesicles (SUV) was collected and the pellet was discarded(FIG. 1 ). To obtain particles with a narrower size distribution, theobtained SUV particles were further extruded through polycarbonatemembrane (30 nm pore size).

The obtained SUVs were further analyzed using dynamic light scattering(DLS) and transmission electron microscopy (TEM) techniques (FIG. 11 ).The final phospholipid concentration in a given sample was determinedvia inductive coupled plasmon mass spectroscopy (ICP-MS). The number ofliposomes in solution and the number of oligonucleotides on the surfaceof a liposome can be calculated according to the equation depicted inFIG. 12 .

Preparation of DNA Functionalized Liposomal SNAs

In order to prepare liposomal SNAs, 15 μM of the desired 3′-tocopherolmodified oligonucleotide was added to a SUV solution (1.3 mM of[phospholipid]) and allowed to shake overnight. The resulting solutionwas then purified via gel filtration chromatography on cross-linkedsepharose column (Separose CL 4B, Aldrich). The particle sizedistribution was analyzed using DLS. To observe the liposomal SNAs usingTEM, the samples were placed onto plasma-cleaned carbon TEM grids andfurther stained with solution of uranyl acetate (2% w/v) (stained for 2min then washed with water and allowed to dry). The dried grid was thenimaged under the Hitachi-2300 STEM electron microscope.

Gel Electrophoresis of Liposomal SNAs

All gel electrophoresis experiments were conducted in a 1% agarose gelin 1×TBE (tris borate, EDTA) buffer. The samples were loaded in thewells with the aid of glycerol (30% v/v, 5 μL) as a loading agent. Thegel chamber was filled with 1×TBE and was precooled with ice. The gelswere run at 70V for 1 hour at 10° C. and the images of the gel wererecorded with Fluorchem Q with a Cy5 filter.

Quantification of DNA Density on the Liposomal Surface

To determine the loading of DNA on the surface of liposomes, anincreasing concentration of Cy5-labeled 3′ tocopherol-modified DNA wasincubated overnight with a fixed concentration of SUVs (1.3 mM of [P]).The liposomal SNAs were then analyzed using gel electrophoresis. Toquantify the DNA density functionalized on the SUVs, the constructs weredissolved in 1% SDS solution and absorbance was collected at 260 nm andcalculated using the extinction coefficient of the respective DNAstrand. The number of liposomes in the corresponding solution wascalculated using the theoretical equation with the assumption that thephospholipid concentration of the liposomes remains constant afterfunctionalization.

Melting Assays

A two nanoparticle-component system was formed using liposomal SNAsfunctionalized with strands complementary to the linker strand asdescribed in Table 1. The aggregates were formed by addition of two DNAfunctionalized Liposomal SNAs and hybridizing them to the linker strandin a 1:1 ratio (the total DNA concentration 1.5 μM, volume 1 mL). Theabsorbance spectra for the liposomal SNAs with the linker was collectedusing Cary 5000 UV-Vis spectrometer and compared to the absorbancespectra of liposomal SNAs without the linker. The aggregates were thensubjected to a gradual increase in temperature at a rate of 0.25° C./minfrom 20 to 65° C. and the absorbance was monitored at 260 nm for theaggregates.

Rhodamine Encapsulation

Dry DOPC monomer (25 mg) was resuspended in a 20 mM Sulforhodamine Bsolution in HBS (5 mL). The resulting suspension was gradually extrudedthrough a series of polycarbonate membranes, 100 nm, 80 nm, 50 nm, 30 nmsizes. The rhodamine containing liposomes were separated from the freerhodamine via gel filtration chromatography on cross-linked sepharose(Sepharose CL-4B, Aldrich). The obtained particles were functionalizedwith DNA-tocopherol conjugates using the procedure described above. Toanalyze the serum stability of the constructs, the rhodamine containingliposomes and liposomal-SNAs were suspended in 10% fetal bovine serumsolution in HBS, and the release of the dye was monitored in aFluorlog-3 system by exciting the sample at 420 nm and measuring theintensity at 480 nm.

Cell Culture Studies

SKOV-3 cells were purchased from American Type Culture Collection (ATCC)and were grown in the McCoy's 5A medium with 10% heat inactivated fetalbovine serum, 100 U of penicillin and 50 μg of streptomycin andmaintained at 37° C. with 5% CO2 as per ATCC instructions. For cellularstudies, the cells were plated 24 hours prior to the treatment at the50% confluency.

Confocal Microscopy of Liposomal SNAs

For visualizing of the cellular internalization of liposomal SNAs, theSKOV3 cells were plated on 35 mm FluoroDish™ chamber at 50% confluent.The cells were incubated with Cy5-labeled liposomal SNAs (0.1 μM of DNAconcentration) in media for 20 hours followed by three washes with 1×PBScontaining 0.01% (by volume) tween-20 then replaced with fresh media.The nuclei were stained with Hoechst 3342 (Invitrogen) following themanufacturer's protocol. The live cells were then imaged with Zeiss LSM510 inverted laser scanning confocal microscope with Mai Tai 3308 laser(Spectra-Physics) at 40× magnification. The Hoechst was excited at 780nm and collected at 390-495 nm and excited at 640 nm and emission at650-710 nm.

Flow Cytometry Experiments

To compare the cellular uptake of liposomal SNAs to free-DNA strand, thecells were plated on a 96 well in 100 μL of media and incubated with 0.1μM concentration of free-DNA or liposomal SNAs and for 24 hours. Theuntreated cells were used as a negative control for the experiment.After the incubation period, the cells were washed 3 times with 1×PBScontaining 0.01% (by volume) of Tween-20 and then trypsinized to form asuspension. Flow cytometry was performed on the cellular suspensionusing Cy5 intensity channel on Guava easyCyte 8HT (Millipore, USA) usingthe signal from the untreated cells as for background intensity. Theerror values were calculated using the standard error of the mean ofmedian signal from different wells representing a single sample.

Cytotoxicity Studies (MTT Assay)

To evaluate the cytotoxicity of the liposomal SNAs, the SKOV-3 cellswere plated on a 96 well 24 hours before the experiment. The cells weretreated with liposomal SNAs at varying concentration of DNA for 24hours. The cytotoxicity of liposomal SNAs was compared to DharmaFECT® 1(Dharmacon), a commercially available transfection agent. The cells weretransfected with varying concentrations of DNA transfected withDharmaFECT® 1 following the manufacturer's transfection protocol. Thecells, that didn't receive the treatment, were used as a negativecontrol. After the incubation period of 24 hours, the cells were washedthree times with 1×PBS and incubated with alamarBlue® solution (ThermoFisher Scientific Inc.) and incubated at 37° C. in 5% CO2 for 4 hours.The fluorescence emission at 590 nm was recorded using the BioTek,Synergy H4 Hybrid Reader.

Western Blotting to Quantify HER2 Protein Knockdown

The SKOV-3 cells were plated in a 6-well plate and incubated at 37° C.in 5% CO2 overnight. The cells were incubated with anti-HER2 antisenseliposomal SNAs and scrambled liposomal SNAs. After a treatment of 24hours, the medium was replaced with fresh medium and the cells wereallowed to grow for an additional 48 hours. To analyze the HER2 proteinknockdown, the cells were collected and re-suspended in 100 μL ofmammalian cell lysis buffer (Cell Signaling, MA, USA) containingprotease and phosphatase inhibitors. (Thermo Scientific, IL, USA). Theprotein concentration in the cell lysates was determined using a BCAProtein Assay Kit (Pierce, IL, USA). Equal amounts (20 μg) of proteinswere fractionated by 4-20% Precast gradient gel (Bio-Rad) andtransferred to a nitrocellulose membranes (Thermo Scientific, IL, USA).The membrane was blocked using 5% dry non-fat milk solution (w/v) intris-buffered saline (TBS). The proteins were detected with primaryrabbit antibodies against HER2 (1:1000), and GAPDH (1:500) followed byanti-rabbit secondary antibodies (1:10,000) (LI-COR Biosciences, NE,USA). The fluorescence signal was recorded using the Odyssey® InfraredImaging System (LI-COR Biosciences, NE, USA).

Synthesis

A typical liposomal SNA was synthesized in two steps (FIG. 7 ). Thefirst step involves the preparation of 30 nm diameter unilamellarvesicles from lipid monomers. This size particle is ideal from thestandpoints of SNA transfection and is in the appropriate range formaximizing higher blood circulation and minimizing clearance through thekidneys. Unfortunately, liposomes in this size regime are often unstableand fuse to form larger structures. Therefore, a goal of this work wasto determine a way of synthesizing such structures and avoiding suchparticle growth pathways.

To prepare small unilamellar vesicles (SUVs), DOPC(1,2-dioleoyl-sn-glycero-3-phosphocholine), an unsaturated lipidcontaining two oleic acid derivatives extending from a phosphate moietyand terminated with a quaternary ammonium head group, was selected. In atypical experiment, a suspension of DOPC monomers in 20 mM HBS wassonicated to produce on average 30 nm SUV particles. The particles wereisolated by centrifugation (100,000×g). Further extrusion of thismaterial, through a polycarbonate membrane with 30 nm pores yieldedparticles with a polydispersity index (PDI) of 0.11 in 70% overallyield. The particles were then redispersed in saline, and DLS was usedto confirm their 30±3 nm diameter, which was also subsequently confirmedby TEM analysis using negative staining.

The second step of the synthesis involves surface functionalization ofthe liposome with a nucleic acid derivative possessing a hydrophobictocopherol moiety, which effectively inserts into the lipid bilayerdefining the SUV. Although a variety of hydrophobic head groups might besuitable [Pfeiffer et al., J. Am. Chem. Soc. 126: 10224 (2004);Banchelli et al., J. Phys. Chem. B 112: 10942 (2008); Dave et al., ACSNano 5: 1304 (2011); Jakobsen et al., Bioconjugate Chem. 24: 1485(2013)], α-tocopherol (a form of vitamin E) was chosen because of itsbiocompatibility and low cost. The α-tocopherol was installed ontonucleic acid strands (DNA) via a conventional oligonucleotide synthesis,utilizing a commercially available tocopherol phosphoramidite derivative(Glenn Research). The liposomal SNAs were synthesized by incubating asuspension of SUVs (1.3 mM by lipid) with the nucleic acid-tocopherolconjugates (16 mM) using a lipid-to-nucleic acid ratio of 8:1 for 12hours at room temperature. The liposome-free tocopherol-nucleic acid wasthen removed from the sample by size exclusion chromatography on asepharose column (Sepharose 4LB). In the case of DNA, a significant dropin the zeta potential from −1 to −23 occurs after this step, indicatingliposome surface functionalization with the negatively charged nucleicacid. In addition, dynamic light scattering (DLS) analysis of the finalnanoparticle samples showed an increase in particle size from 30 to 46nm, consistent with the loading of the 8-9 nm long duplex structure. Todetermine quantitatively the average number of nucleic strands loadedonto the surface of a liposome, the liposomal-SNAs were dissolved in thepresence of Triton X to release them. The final nucleic acidconcentration was determined by measuring the absorbance at 260 nmrelative to a calibrated oligonucleotide standard. The liposomal SNAscoated with DNA had on average of 70 strands per particle (FIG. 17 ).This density is lower than a typical gold-based SNA structure [Hurst etal., Anal. Chem. 78:8313 (2006)] but sufficient to exhibit many of thecooperative properties of such structures. A graphical depiction of aliposomal SNA is provided in FIG. 16 .

These liposome SNA structures have several interesting properties.First, they are remarkably stable compared to the native 30 nm liposomeconstructs from which they have been derived (FIG. 2 , FIG. 8 ). Forexample, if the SUVs without an oligonucleotide surface layer are storedfor four days at 37° C. (physiological temperature), they fuse and formlarger polydisperse structures (on average 100 nm structures with somemicron-sized entities). In contrast, the liposomal SNAs show no evidenceof particle degradation or fusion over the same time period under nearlyidentical conditions. This increase in stability for the liposomal-SNAsystem is likely a result of the repulsive forces between the negativelycharged nucleic acid strands that comprise the liposomal-SNAs surface,which both stabilizes the individual particles and inhibitparticle-particle fusion interactions [Li et al., Bioconjugate Chem. 24:1790 (2013)]. Moreover, the negatively charged DNA corona on theliposomal-SNA serves as a protecting layer for the liposomal corepreventing its degradation in the presence of serum proteins [Senior etal., Life Sci. 30: 2123 (1982); Kim et al., Arch. Pharmacal Res. 14: 336(1991); Sulkowski et al., J. Mol. Struct. 744-747: 737 (2005)]. Forexample, serum stability of the liposomal-SNAs system was investigatedby measuring the release of a sulforhodamine dye physically incorporatedwithin the core of a liposomal-SNA at a self-quenching concentration of20 mM (core concentration). In this experiment, rupture of the liposomalcore results in a release of the sulforhodamine dye from the interior ofthe particle and a subsequent elimination of self-quenching, thus givingrise to an increase in fluorescence [Versluis et al., J. Am. Chem. Soc.135: 8057 (2013)]. In a typical experiment, rhodamine-containingliposomal nanoparticles were incubated in 10% fetal bovine serum at 37°C., and the fluorescence spectra were recorded continuously for 3 hours.The same stability study was performed for non-functionalized particles.Similar to the thermal stability studies, DNA-functionalized particlesremained stable in serum for the duration of experiment. No release ofthe dye was observed during 3 hours of incubation. In contrast,incubation of the bare DOPC liposomes led to a significant release ofthe rhodamine fluorophore indicating fast decomposition of the liposomalstructure in serum (FIG. 8 ).

A second property of liposomal SNAs is their ability to cooperativelybind complementary nucleic acids. This is a hallmark feature of all SNAsand derives from the densely packed and highly oriented configuration ofthe surface-bound nucleic acids. To explore the binding and subsequentmelting properties of the liposomal-SNA constructs, two sets ofliposomal-SNA nanoparticles were synthesized, each made with differentDNA sequences: particle A and particle B. A DNA linker sequence that iscomplementary to the oligonucleotide sequences of the liposomal-SNAs wasused to facilitate polymerization through hybridization. Upon additionof the linker sequence to an equimolar mixture of the two liposomal SNAparticles, aggregation occurred as evidenced by DLS and eventually aflaky precipitate was formed [Dave et al., ACS Nano 5: 1304 (2011)].These aggregates were re-suspended in 20 mM HBS (150 mM NaCl), and amelting analysis was performed by monitoring the absorbance at 260 nm.Importantly, a remarkably narrow melting transition was observed at47.5° C. (full width at half-maximum of the first derivative isapproximately 2° C.), which is highly diagnostic of an SNA structurewith a high surface density of nucleic acids (FIG. 9 ).

An important property of SNAs pertains to their ability to enter cellswithout the need for ancillary transfection agents [Cutler et al., J.Am. Chem. Soc. 134: 1376 (2012)]. To determine if liposomal SNAs exhibitthis behavior, ovarian cancer ascites (SKOV3, American Type CultureCollection) were incubated in the presence of the liposomal SNAssynthesized with a 5′-Cy5-labeled DNA in the absence of any transfectionagents at different DNA concentrations. The uptake of liposomal-SNAs inSKOV3 cells was analyzed using confocal microscopy and flow cytometrytechniques. Remarkably, liposomal SNAs readily entered cells in highquantities even after 1 hour of incubation, which demonstrates theirutility as intracellular probes and target regulating agents. Inaddition, no significant uptake of free DNA strand (5′-Cy5-labeled) inSKOV3 cells was detected even after 36 hours of incubation underidentical conditions. Similar to the Au—SNAs, high uptake ofliposomal-SNAs in SKOV3 cells didn't cause any toxicity even at highconcentrations (FIG. 10 ). Conversely, employment of the DharmaFECT inan attempt to deliver equal DNA delivered by the liposomal-SNAs resultedin a significant cytotoxicity, which reduced cell viability to 35% overa 24 hour time period of incubation.

After establishing that liposomal-SNAs are not cytotoxic, aliposomal-SNA was synthesized that was capable of knocking down humanepidermal growth factor receptor 2 (HER2)—an oncogene overexpressed inSKOV3 cells [Zhang et al., J. Am. Chem. Soc. 134: 16488 (2012)]. Tocompare the effectiveness of the antisense activity of liposomal-SNAs tothat of conventional transfection systems, SKOV3 cells were incubated inthe presence of anti-HER2 liposomal-SNAs, and control liposomal-SNAs(each at a total DNA concentration of 1 μM). After 72 hours ofincubation, the cells were harvested and analyzed for protein content byWestern blotting. Importantly, HER2 protein levels were reduced by 85%in the presence of anti-HER2 liposomal-SNAs compared to the internalreference gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (FIG. 10). Collectively, these results demonstrate the potential to use theliposomal-SNAs to effect both cellular transfection and gene regulation.

In summary, a scalable synthetic route for novel metal-free liposomalSNAs has been developed. Such structures are assembled rapidly fromreadily available, non-toxic starting materials. The nucleic acidarchitecture not only stabilizes these small liposomal structures butalso facilitates their internalization by SKOV3 cells. Consequently,such structures show utility as new biocompatible gene regulationconstructs that exhibit many of the attractive properties of the moreconventional gold nanoparticle-based SNAs.

Example 6—Testing Liposomal Particles in Ramos-Blue™ Cells

Ramos-Blue™ cells are NF-κB/AP-1 reporter B lymphocyte cells. Ramos-Blueis a B lymphocyte cell line that stably expresses anNF-κB/AP-1-inducible SEAP (secreted embryonic alkaline phosphatase)reporter gene. When stimulated, they produce SEAP in the supernatantthat can be readily monitored using the QUANTI-Blue assay. QUANTI-Blueis a SEAP detection medium that turns blue in the presence of SEAP (FIG.14 ).

When contacted with CpG-containing oligonucleotides, activation of theRamos-Blue cells was detected (FIG. 15 ). Representative compounds weresynthesized based on the TLR 9-agonizing oligonucleotide CpG 7909(5′-TCGTCGTTTTGTCGTTTTGTCGTT-3′ (SEQ ID NO: 8)). These include CpG 7909with a phosphodiester backbone densely functionalized on 13 nm goldnanoparticles (CpG 7909-po SNA (Au)), CpG 7909 with a fullyphosphorothioate backbone (CpG 7909-ps), a liposomal SNA with aphosphodiester CpG 7909 (7909 targeting (particle)), a liposomal SNAwith the C and G of the all phosphodiester backbone oligonucleotideinverted to eliminate the TLR 9 binding site (7909 control (particle)),CpG 7909 with a phosphodiester backbone and 3′-tocopherol lipid withoutbeing formulated into a liposomal SNA (7909 targeting (tocopherol)), anda control sequence with the C and G inverted that is also not formulatedinto a liposomal SNA (7909 control (tocopherol)). These compounds wereserially diluted then incubated with Ramos-Blue cells, a cell line thatexpresses secreted alkaline phosphatase (SEAP) upon activation of thepro-inflammatory transcription factor NF-κB, overnight and then probedfor SEAP levels in the cell culture media via the QuantiBlue kit(InVivogen). Activation is measured by absorption of light at 650 nm.

Example 7—Use of Liposomal Particles to Regulate HIF1-α

To further demonstrate the effectiveness of a composition of thedisclosure, liposomal particles were designed to individually targetHIF1-α and BAX. The experiments utilized the Neuro-2a (N2A) cell line,which is a fast-growing mouse neuroblastoma cell line. Contacting theN2A cells with liposomal particles targeting both HIF1-α (FIG. 18 ) andBAX (FIG. 19 ) showed a significant reduction in the amount of targetgene product. In each of the experiments, the relative amount of mRNAexpression was determined by quantitative PCR (qPCR) 72 hours afterbeginning treatment of the N2A cells in 6-well plates—cells were treatedwith the liposomal particles for 24 hours in OptiMEM prior to removal ofthe liposomal particles and replacement of the media with MEM and 10%fetal bovine serum (FBS).

For the experiments in which HIF1-α was targeted, the N2A cells werefirst subjected to Cocl2-stimulated hypoxia, which increased HIF1-α mRNAexpression by about 50%. Next, the N2A cells were contacted with theliposomal particles functionalized with siRNA directed against HIF1-α.The contacting resulted in a knockdown of HIF1-α of about 50% (FIG. 18).

For the experiments in which BAX was targeted, treatment of N2A cellswith the resulted in an approximate 65% knockdown of BAX mRNA by theliposomal particles and greater than 50% knockdown of BAX mRNA by lipidmicelle SNAs (as measured against control liposomal SNAs) (FIG. 19 ).

These experiments showed that the liposomal particles of the disclosureare highly effective at inhibiting target gene expression in mammaliancells.

It will be evident to one skilled in the art that the present inventionis not limited to the foregoing illustrative examples, and that it canbe embodied in other specific forms without departing from the essentialattributes thereof. It is therefore desired that the examples beconsidered in all respects as illustrative and not restrictive,reference being made to the appended claims, rather than to theforegoing examples, and all changes which come within the meaning andrange of equivalency of the claims and therefore intended to be embracedtherein.

What is claimed is:
 1. A liposomal particle having a substantiallyspherical geometry, comprising: a lipid bilayer comprising a pluralityof lipid groups; and single-stranded oligonucleotides that areinhibitory RNA or antisense DNA, wherein all the oligonucleotides areattached to the surface of the liposomal particle, wherein eacholigonucleotide is an oligonucleotide-lipid conjugate containing onelipophilic tethered group anchored into the external side of the lipidbilayer, wherein one or more of the oligonucleotides hybridize to a genecomprising a polynucleotide encoding a gene product and arecomplementary to all or a portion of the polynucleotide, wherein thehybridizing between the oligonucleotide and the polynucleotide occursover a length of the polynucleotide with a degree of complementaritysufficient to inhibit expression of the gene product, and wherein thediameter of the liposomal particle is less than or equal to 50nanometers (nm).
 2. The liposomal particle of claim 1, wherein theplurality of lipid groups comprises a lipid selected from the groupconsisting of the phosphatidylcholine, phosphatidylglycerol, andphosphatidylethanolamine family of lipids.
 3. The liposomal particle ofclaim 2, wherein the lipid is selected from the group consisting of1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1,2-dimyristoyl-sn-phosphatidylcholine (DMPC),l-palmitoyl-2-oleoyl-sn-phosphatidylcholine (POPC), 1,2-distearoyl-sn-glycero-3-phospho-(l′-rac-glycerol) (DSPG),1,2-dioleoyl-sn-glycero-3-phospho-(I′-rac-glycerol) (DOPG),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), and1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DPPE).
 4. Theliposomal particle of claim 1, wherein the lipophilic tethered groupcomprises a tocopherol or cholesterol.
 5. The liposomal particle ofclaim 4, wherein the tocopherol is chosen from the group consisting of atocopherol derivative, alpha-tocopherol, beta-tocopherol, gamma-tocopherol and delta-tocopherol.
 6. The liposomal particle of claim1, wherein the inhibitory RNA is selected from the group consisting of asmall inhibitory RNA (siRNA), a miRNA, an inhibitory RNA that forms atriplex with double stranded DNA, and a ribozyme.
 7. The liposomalparticle of claim 1, wherein the liposomal particle comprises from about10 to about 80 oligonucleotides.
 8. The liposomal particle of claim 7,wherein the particle comprises 70 oligonucleotides.
 9. The liposomalparticle of claim 7, wherein the liposomal particle comprises 30oligonucleotides.
 10. The liposomal particle of claim 1, wherein theoligonucleotides are modified oligonucleotides having phosphorothioatelinkages.
 11. The liposomal particle of claim 1, wherein the diameter ofthe liposomal particle is less than or equal to 40 nanometers (nm). 12.The liposomal particle of claim 1, wherein the diameter of the liposomalparticle is less than or equal to 35 nanometers (nm).
 13. The liposomalparticle of claim 1, wherein the diameter of the liposomal particle isless than or equal to 30 nanometers (nm).
 14. The liposomal particle ofclaim 1, wherein the lipophilic tethered group is at the 3′-end of eacholigonucleotide.
 15. A plurality of liposomal particles comprising aplurality of the liposomal particle of claim 1, wherein the plurality ofliposomal particles has a mean diameter of less than or equal to about50 nanometers (nm).
 16. The plurality of liposomal particles of claim15, wherein the plurality of liposomal particles has a mean diameter ofless than or equal to about 40 nanometers (nm).
 17. The plurality ofliposomal particles of claim 15, wherein the plurality of liposomalparticles has a mean diameter of less than or equal to about 35nanometers (nm).
 18. The plurality of liposomal particles of claim 15,wherein the plurality of liposomal particles has a mean diameter of lessthan or equal to about 30 nanometers (nm).
 19. A liposomal particlehaving a substantially spherical geometry, consisting of: a lipidbilayer consisting of a plurality of lipid groups; and single-strandedoligonucleotides that are inhibitory RNA or antisense DNA, wherein allthe oligonucleotides are attached to the surface of the liposomalparticle, wherein each oligonucleotide is an oligonucleotide-lipidconjugate containing one lipophilic tethered group anchored into theexternal side of the lipid bilayer, wherein one or more of theoligonucleotides hybridize to a gene comprising a polynucleotideencoding a gene product and are complementary to all or a portion of thepolynucleotide, wherein the hybridizing between the polynucleotide andthe oligonucleotide occurs over a length of the polynucleotide with adegree of complementarity sufficient to inhibit expression of the geneproduct, and wherein the diameter of the liposomal particle is less thanor equal to 50 nanometers (nm).
 20. A plurality of liposomal particlescomprising a plurality of the liposomal particle of claim 19, whereinthe plurality of liposomal particles has a mean diameter of less than orequal to about 50 nanometers (nm).
 21. The plurality of liposomalparticles of claim 20, wherein the plurality of liposomal particles hasa mean diameter of less than or equal to about 40 nanometers (nm). 22.The plurality of liposomal particles of claim 20, wherein the pluralityof liposomal particles has a mean diameter of less than or equal toabout 35 nanometers (nm).
 23. The plurality of liposomal particles ofclaim 20, wherein the plurality of liposomal particles has a meandiameter of less than or equal to about 30 nanometers (nm).